ES2664992T3 - Modified Single Chain Polynucleotide - Google Patents

Abstract

A polynucleotide or salt thereof, the polynucleotide comprising a polynucleotide with a direct sense strand corresponding to a target gene, and a polynucleotide with an opposite strand having a polynucleotide sequence complementary to that of the direct sense strand polynucleotide and having a structure represented by the following formula, in which the 5 'end of the opposite strand polynucleotide and the 3' end of the direct strand polynucleotide are linked by a linker through phosphodiester bonds: ** ( See formula) ** in which p represents an integer from 0 to 4, q represents an integer from 4 to 10, L5 represents a simple bond or -O-, L6 represents - (C> = O) -NH- or -NH- (C> = O) - starting from the link with (CH2) p, L5 is attached to the benzene ring at the meta or para position, and provided that if L5 is -O-, then, p represents a whole number from 1 to 4.

Description

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DESCRIPTION

Modified single stranded polynucleotide Technical field

The present invention relates to a single stranded polynucleotide that has an RNA interference effect and / or a gene expression inhibitory effect, to the use of the polynucleotide, to a method of producing the polynucleotide, to a pharmaceutical composition comprising the polynucleotide, etc. .

Background Technique

Procedures for inhibiting the expression of a target gene in cells, tissues or individuals include an approach in which double stranded RNA is introduced into cells, tissues or individuals. Through this introduction of double stranded RNA, the mRNA that has homology to the sequence is degraded, so that expression of the target gene is inhibited. This effect is called "RNA interference" or "RNAi". RNA interference was initially reported in C. elegans (see for example, Non-Patent Reference 1) and subsequently also reported in plants (see for example, Non-Patent Reference 2).

It has been reported that double stranded RNA consisting of 21 nucleotide strands of direct and opposite direction having a 2 nucleotide protrusion at the 3 'end (small interfering RNA: siRNA) has an RNA interference effect on cells Cultivated vertebrates (see for example, Non-Patent Reference 3). The siRNA is considered useful for the identification of gene functions, screening of suitable cell strains for the production of useful substances, regulation of genes involved in diseases, etc., but, nevertheless, in a characteristic way, it is easily degraded with RNase ( see for example, Non-patent Reference 4).

It has been reported that a double stranded polynucleotide having alternately combined DNA and 2'-OMeRNA nucleotide units, instead of the RNAs constituting the siRNA, is resistant to the RNAse and has an RNA interference effect (see for example, Non-patent Reference 1).

Some reports have been made on the modification of the 5 'ends of the strands of direct and opposite directions in the siRNA. It has been reported that the siRNA having a 6-aminohexyl phosphate group at the 5 'end of the opposite or opposite direction strand has an inhibitory effect against the expression of the target mRNA (see for example, Non-Patent Reference 5 ). On the other hand, it has been reported that the siRNA having a 6-aminohexyl phosphate group at the 5 'end of the opposite strand has no inhibitory activity against expression of the target mRNA (see for example, Non-patent Reference 6) . It has also been reported that the siRNA that has a 3-aminohexyl phosphate group at the 5 'end of the direct sense strand has an inhibitory effect against the expression of the target mRNA, while the siRNA that has a 3-aminohexyl phosphate group in the 5 'end of the opposite strand has no inhibitory activity against expression of the target mRNA (see for example, Non-Patent Reference 7). It has been reported that inhibitory activity against expression of the target mRNA is observed lower in the siRNA having the 6-aminohexylphosphate group or the 3-aminopropyl phosphate group at the 5 'end of the opposite strand than in the unmodified siRNA , but not completely lost. (see for example, Non-patent Reference 8.

It has been reported that the siRNA having fluorescein at the 5 'end of the opposite or opposite direction strand also has an inhibitory effect against expression of the target mRNA (see for example, Non-patent Reference 9). It has been reported that siRNAs having a steroid or lipid structure at the 5 ′ end of the direct or opposite direction strand, the siRNA having a steroid or lipid structure at the 5 ′ end of the detected sense strand it has inhibitory activity against expression of the target mRNA (see for example, Non-Patent Reference 8). It has been reported that, when the siRNA has an ortho-nitrobenzyl derivative, which can be removed by UV irradiation, at the 5 'end of the opposite strand, its inhibitory activity against expression of the target mRNA can be controlled using the UV irradiation (see for example, Non-Patent Reference 10).

The siRNA whose 3 'end of the direct sense strand and the 5' end of the opposite direction strand are joined by a loop consisting of approximately 4 nucleotide units forms a single stranded polynucleotide called short hairpin RNA (shRNA). It has been shown that the shRNA that has a 19 bp stem residue on the rest of the stem has less activity than a 19 bp siRNA that has the same nucleotide sequence as the previous one (see for example, Non-Patent Reference 9). Although the mRNA comprising the 19 bp stem and the loop having two nucleotides is replaced by a non-nucleotide linker such as propyl phosphate units was synthesized and examined to determine its inhibitory activity against expression of the target mRNA, it was not observed. improvement in activity, compared to unmodified siRNA (see for example, Non-Patent Reference 9). An example using an ortho-nitrobenzyl derivative has been reported as an siRNA in which the 3 'end of the direct sense strand and the 5' end of the opposite direction strand are joined by a non-nucleotide linker (see for example , Non-patent Reference 8). This 19 bp siRNA whose strands of direct and opposite direction are linked by ortho-nitrobenzyl derivative has a lower inhibitory activity against mRNA expression than unmodified siRNA. In addition, cultured cells transfected with this siRNA were irradiated with UV for 10 minutes and examined for

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siRNA inhibitory activity against expression of the target mRNA. As a result, the inhibitory activity of siRNA against expression of the target mRNA was lower than that of unmodified siRNA. A single stranded polynucleotide having a structure in which the 5 'end of the opposite direction strand and the 3' end of the direct sense strand are joined by a linker containing a phenyl group to form a phosphodiester structure in each of These extremes are not yet known. EP2233573 analyzes the con of the zipper interference RNA (RNAi).

X-ray analysis of a complex of a strand in the opposite direction with the Argonauta (Ago) protein known to participate in RNAi activity has shown that the phosphate group at the 5 'end of the strand in the opposite direction and its neighboring nucleotides are strongly bound to the Ago PIWI domain (see for example, Non-Patent Reference 11). It has been reported that after the introduction of chemically synthesized siRNA into cells, both the direct sense and the opposite sense strands are phosphorylated at their 5 'end (see for example, Non-patent Reference 12). In human cells, it has been reported that hClp1 RNA kinase is responsible for the 5 'phosphorylation of the siRNA (see for example, Non-Patent Reference 13). When the siRNA with the 5 ′ phosphorylated end and the siRNA having a 5 ′ non-phosphorylated end are introduced separately into cells, and their RNAi activities are compared, no difference in activity between them is observed, which indicates that the siRNA having a 5 'non-phosphorylated end is readily subjected to phosphorylation in the cells (see for example, Non-Patent Reference 9).

In the case of using the rRNA in which the 3 'end of the direct sense strand and the 5' end of the opposite strand are joined by a loop, this shRNA is cleaved intracellularly by the Dicer protein or an endonuclease to forming a counter-strand having a phosphate group at its 5 'end (see for example, Non-patent Reference 9). It cannot be expected that RNAsh comprising a 19 bp stem and a loop having two nucleotides substituted by propyl phosphate units undergo intracellular dicer or endonuclease excision, because propyl phosphate units are nuclease resistant (see for example, Reference non-patent 9). Alternatively, it can be expected that the shRNA comprising a 19 bp stem and a loop having an ortho-nitrobenzyl derivative form a counter-strand that has a phosphate group at the 5 'end by UV irradiation. Such UV irradiation, however, is difficult to apply to living organisms due to possible adverse reactions and due to the difficulty of applying UV irradiation inside a living organism (see for example, Non-patent Reference 8). A single stranded polynucleotide comprising a stem of 19 bp or less and a loop having a non-nucleotide linker alone, which is cleaved intracellularly by Dicer or endonuclease without UV irradiation to form a counter-strand that has a phosphate group at the 5-terminus. ', is not yet known.

The present inventors have carried out careful studies to obtain a polynucleotide that has an effect of RNA interference and / or an inhibitory effect of gene expression and, consequently, they have completed the present invention by discovering a single stranded polynucleotide having an effect of RNA interference and / or a gene expression inhibitory effect, which is derived from a double-stranded polynucleotide comprising a direct sense strand corresponding to a target gene, and a counter-stranded polynucleotide having a complementary nucleotide sequence of the polynucleotide with a direct sense strand, and has a structure in which the 5 'end of the opposite direction strand and the 3' end of the direct sense strand are joined by a linker containing a phenyl group to form a phosphodiester structure at each of these extremes.

Reference

Patent references

Patent reference 1: International Publication No. WO 2010/001909 Non-patent references

Non-patent reference 1: Nature, 1998, Vol. 391, p. 806-811 Non-patent reference 2: Science, 1999, Vol. 286, p. 950-952 Non-patent reference 3: Nature, 2001, Vol. 411, p. 494-498 Non-patent reference 4: Clinical Chemistry, 2002, Vol. 48, p. 1647-1653 Non-patent reference 5: Molecular Cell, 2002, Vol. 10, p. 537-548 Non-patent reference 6: Nucleic Acids Research, 2003, Vol. 31, p. 2705-2716 Non-patent reference 7: Molecular Cell, 2002, Vol. 10, p. 549-561 Non-patent reference 8: Oligonucleotides, 2007, Vol. 17, p. 35-43

Non-patent reference 9: Antisense Nucleic Acid Drug Development, 2003, Vol. 13, p. 83-105 Non-patent reference 10: Biochimica Biophysica Acta, 2006, Vol. 1758, p. 394-403 Non-patent reference 11: Nature, 2005, Vol. 434, p. 663-666 Non-patent reference 12: Cell, 2001, Vol. 107, p. 309-321 Non-patent reference 13: Nature, 2007, Vol. 447, p. 222-226

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Summary of the invention

Problems that the invention will solve

An object of the present invention is to provide a polynucleotide having an effect of RNA interference and / or an inhibitory effect of gene expression.

A further object of the present invention is to provide a polynucleotide that is resistant to RNase and that has an effect of RNA interference and / or an inhibitory effect of gene expression.

A further object of the present invention is to provide a method for inhibiting gene expression using the polynucleotide.

A further object of the present invention is to provide a pharmaceutical composition comprising the polynucleotide.

Means for solving problems Specifically, the present invention consists of:

(1) A polynucleotide or salt thereof, the polynucleotide comprising a polynucleotide with a direct sense strand corresponding to a target gene, and a polynucleotide with an opposite direction strand having a polynucleotide sequence complementary to that of the polynucleotide with strand of direct sense and having a structure represented by the following formula, in which the 5 'end of the polynucleotide with strand in the opposite direction and the 3' end of the polynucleotide with strand in the direct direction are joined by a linker through links phosphodiester:

[Formula 2]

image 1

p represents an integer from 0 to 4, q represents an integer from 4 to 10,

L2 3 4 5 represents a simple bond or -O-,

L6 represents - (C = O) -NH- or -NH- (C = O) - starting from the link with (CH2) p,

L5 is attached to the benzene ring in the meta or para position, and provided that L5 is -O-, then, p represents an integer from 1 to 4.

(2) The polynucleotide or salt thereof according to (1), in which the sum of pyq is an integer of 4 or greater, L5 is a simple bond, L6 is - (C = O) -NH- , and L5 is attached to the benzene ring in the para position, preferably in which the sum of pyq is an integer of 8 or greater.

(3) The polynucleotide or salt thereof according to (1), in which p is 0 or 2, q is an integer of 6 or greater, L5 is a single bond, L6 is - (C = O) -NH-, and L5 is attached to the benzene ring in the position for, preferably where q is 6 or 8 more preferably where q is 8.

(4) The polynucleotide or salt thereof according to (1), where p is 2, q is 8, L5 is a single bond, L6 is - (C = O) -NH-, and L5 is bound to the benzene ring in the position for.

(5) A polynucleotide or salt thereof, the polynucleotide comprising a polynucleotide with a direct sense strand corresponding to a target gene, and a polynucleotide with an opposite direction strand having a polynucleotide sequence complementary to that of the polynucleotide with strand of direct sense, in

which the 5 'end of the strand polynucleotide in the opposite direction and the 3' end of the strand polynucleotide

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Directly, they are connected by means of a linker to form a phosphodiester structure at any of said ends, the linker having a structure represented by the following formula:

[Formula 1]

image2

in which

the oxygen atom attached to the phenyl group is attached to the 5 'end of the strand in the opposite direction to form a phosphodiester structure;

any one of R1, R2, and R3 represents a structure represented by the following formula:

L1- (CH2) m-L2-L3- (CH2CH20) n1- (CH2) n2-0 ^

in which

m represents an integer from 0 to 4, n1 represents an integer from 0 to 4, n2 represents 0 or an integer from 2 to 10,

L1 represents a simple link or -O-,

L2 represents a single bond or -CH (-NH-L4-R) -,

L3 represents a simple link, - (C = O) -NH-, or -NH- (C = O) - starting from the link with L2, provided that L3 is not a single link, then n2 represents an integer number of 2 to 10, provided that each L1 and L2 are a single bond, m is 1, and each of n1 and n2 is 0, then L3- O ^ represents -CH (COOH) NH- (amino acid residue) j-Ser,

-CH (COOH) NH- (amino acid residue) j-Thr,

-CH (NH2) CO- (amino acid residue) j-Ser, or -CH (NH2) CO- (amino acid residue) j-Thr, in which

the rest of the hydroxy group of this serine or threonine is attached to the phosphate group of the 3 'end of the

strand polynucleotide directly, and the amino group of the serine or threonine may be

additionally substituted by an acyl group, j represents an integer from 0 to 2,

L4 represents a single bond, a methylene group, a polymethylene group having 2 to 4 carbon atoms, or the structure - (C = O) -CH2-CH2- (C = O) -O- in which a group Carbonyl of the structure - (C = O) -CH2- CH2- (C = O) -O- is attached to the amino group on the left end of the structural formula to form the structure -NH- (C = O) -CH2- CH2- (C = O) -O- and

R represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, a group of

saturated or unsaturated carbonyl hydrocarbon having from 2 to 30 carbon atoms, or a group

saturated or unsaturated hydrocarbonoxycarbonyl having from 2 to 30 carbon atoms; and the remaining two of R1, R2, and R3 each independently represent a group selected from the group consisting of a hydrogen atom,

an alkyl group having 1 to 8 carbon atoms that a substituent may have, an alkoxy group having 1 to 8 carbon atoms that a substituent may have, a halogen atom,

an alkylcarbonylamino group having an alkyl group having 1 to 9 carbon atoms, and

an alkylcarbonyl group containing an alkyl group having 1 to 8 carbon atoms that may have

a substituent; 6 7 8 9 10

(6) The polynucleotide or a salt thereof according to (5), wherein each R1 and R3 is a hydrogen atom;

(7) The polynucleotide or salt thereof according to (6), in which each L1 and L2 is a single bond, L3 is - (C = O) -NH-, and the sum of my n2 is a number integer of 3 or greater, preferably 8 or greater;

(8) The polynucleotide or salt thereof according to (6), in which each L1 and L2 is a single bond, L3 is - (C = O) -NH-, m is 0 or 2, and n2 is an integer of 6 or greater, preferably 6 or 8, more preferably 8;

(9) The polynucleotide or salt thereof according to (5), in which each R1 and R3 is a hydrogen atom, each L1 and L2 is a single bond, L3 is - (C = O) -NH- , m is 2, and n2 is 8;

(10) The polynucleotide or a salt thereof according to (1) to (9), in which the direct sense strand polynucleotide consists of a polynucleotide represented by the following formula (II), the sense strand polynucleotide opposite consists of a polynucleotide represented by the following formula (III), and the

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Polynucleotide also has the following characteristics of (a) to (d):

5 (y-p)? -Y-'k - 3 '(II)

Y

5’-p- (y-p) 9 - ^ - 3 ’(ni),

(a) and represents an RNA, p represents a 2'-OMeRNA, and each A and u represents a DNA;

(b) t and u represent, identically or differently, any integer from 0 to 5;

(c) (Y-P) 9-Y in the polynucleotide represented by formula (II) has a nucleotide sequence identical to that of the target gene; Y

(d) (Y-P) 9-Y in formula (II) and p- (Y-p) 9 in formula (III) have complementary nucleotide sequences to each other;

(11) The polynucleotide or a salt thereof according to (1) to (9), in which the straight-stranded polynucleotide consists of a polynucleotide represented by the following formula (IV), the strand-shaped polynucleotide The opposite consists of a polynucleotide represented by the following formula (V), and the polynucleotide also has the following characteristics of (a) to (d):

5 1 - (a-p) 3-ai:, - V-3 ’(IV)

Y

5 ’-6, - (a-p) í-Uu-3’ (V),

(a) a and p represent, differently, a DNA or a 2'-OMeRNA, 8 and A represent, identically or differently, a DNA or a 2'-OMeRNA, and u represents, in an identical or different way, any nucleotide selected from a DNA, an RNA, and a 2'-OMeRNA;

(b) p represents an integer of 0 or 1, t is 0 when p is 0 and represents any integer from 0 to 5 when p is 1, s represents any integer of 0 or 1, and u represents any integer of 0 to 5;

(c) (a-p) 9-ap in the polynucleotide represented by formula (IV) has a nucleotide sequence identical to that of the target gene; Y

(d) (a-p) 9 in formula (IV) and (a-p) 9 in formula (V) have nucleotide sequence complementary to each other;

(12) The polynucleotide or a salt thereof according to (1) to (9), in which the straight-stranded polynucleotide consists of a polynucleotide represented by the following formula (VI), the strand-shaped polynucleotide The opposite consists of a polynucleotide represented by the following formula (VII), and the polynucleotide also has the following characteristics of (a) to (d):

image3

Y

5 ’-So- (a-p) 8- (a-p) -ou-3 '(Vil),

(a) a and p represent, differently, a DNA or a 2'-OMeRNA, 8 and A represent, identically or differently, a DNA or a 2'-OMeRNA, and u represents, in an identical or different way, any nucleotide selected from a DNA, an RNA, and a 2'-OMeRNA;

(b) p represents an integer of 0 or 1, t is 0 when p is 0 and represents any integer from 0 to 5 when p is 1, s represents any integer of 0 or 1, and u represents any integer of 0 to 5;

(c) p- (a-p) 8-ap in the polynucleotide represented by formula (VI) has a nucleotide sequence identical to that of the target gene; Y

(d) (a-p) 8 in formula (VI) and (a-p) 8 in formula (VII) have nucleotide sequences complementary to each other;

(13) The polynucleotide or a salt thereof according to (11) or (12), wherein a is a DNA, and p is a 2'-OMeRNA;

(14) The polynucleotide or a salt thereof according to any one of (10) to (13), in which At and Uu are, identically or differently, any of: DNA having a thymine base, a base adenine, or a guanine base; or 2'-OMeRNA having a uracil base, an adenine base, or a guanine base;

(15) The polynucleotide or a salt thereof according to any one of (10) to (14), in which t is 0, and u is

2;

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(16) The polynucleotide or a salt thereof according to any one of (11) to (14), wherein p and t are 0, s is 1, and u is 2;

(17) The polynucleotide or a salt thereof according to (11) to (14), wherein p and t are 0, s is 0 or 1, or is 2, and U2 is a DNA or a 2'-OMeRNA;

(18) The polynucleotide or a salt thereof according to any one of (1) to (9), in which the straight-stranded polynucleotide consists of a polynucleotide represented by the following formula (VIII), the polynucleotide with The opposite thread consists of a polynucleotide represented by the following formula (IX), and the polynucleotide also has the following characteristics of (a) to (c):

5 ’- (a-p) g-3 '(VIII)

Y

5r-p- (a-p) 9- (a-p) -3 ’(IX),

(a) a is a DNA, and p is a 2'-OMeRNA;

(b) p- (a-p) g in the polynucleotide represented by formula (IX) has a nucleotide sequence complementary to that of the target gene; Y

(c) (a-p) g in the formula (VIII) and (a-p) g in the formula (IX) have complementary nucleotide sequences to each other;

(19) The polynucleotide or salt thereof according to any one of (10) to (18), in which any or all of the 2'-OMeRNA residues 1 to 4 are substituted by an ENA or a 2 ', 4 '-BNA / LNA;

(20) The polynucleotide or a salt thereof according to any one of (10) to (19), in which any or all DNA residues 1 to 4 are substituted by an RNA, an ENA or a 2 ', 4'-BNA / LnA;

(21) The polynucleotide or a salt thereof according to any one of (1) to (20), wherein the nucleotides are linked together by a phosphodiester bond or a phosphorothioate bond;

(22) A pharmaceutical composition comprising a polynucleotide or a salt thereof according to any one of (1) to (21) as the active ingredient.

(23) The pharmaceutical composition according to (22), wherein the pharmaceutical composition is for the treatment of a disease derived from gene expression;

(24) a polynucleotide or salt thereof selected from (1) to (21) for use in inhibiting the expression of a target gene in a mammal;

(25) A compound represented by the formula (X) or a salt thereof:

[Formula 3]

image4

wherein Tr represents a hydroxy group protecting group; p represents an integer from 0 to 4; x represents an integer from 4 to 10; L5 represents a simple bond or -O-; L6 represents - (C = O) -NH- or -NH- (C = O) - starting from the bond with (CH2V; and L5 is attached to the benzene ring in the meta or para position; and it is characterized in that the compound It is not

image5

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where n is 4, 6 or 8;

image6

where n is 3, 5 or 7;

image7

where n is 3, 5 or 7; or

image8

where n is 3, 5 or 7.

(26) The compound or salt thereof according to (25), wherein Tr is a 4-methoxytrityl group, a 4,4'-dimethoxytrityl group, a pixyl group, a trityl group, a levulinyl group, or a bis (trimethylsilyloxy) (cyclohexyloxy) silyl group;

(27) The compound or salt thereof according to (25), in which Tr is a 4-methoxytrityl group or a 4,4'-dimethoxytrityl group, the sum of pyq is an integer of 4 or greater, L5 is a single bond, L6 is - (C = O) -NH-, and L5 is attached to the benzene ring in the para position, preferably where the sum of pyq is an integer of 8 or greater;

(28) The compound or salt thereof according to (25), in which Tr is a 4-methoxytrityl group or a 4,4'-dimethoxytrityl group, p is 0 or 2, which is an integer of 6 or greater, L5 is a simple bond, L6 is - (C = O) -NH-, and

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L5 is attached to the benzene ring in the position for, preferably where q is 6 or 8;

(29) The compound or salt thereof according to (25), wherein Tr is a 4-methoxytrityl group or a 4,4'-dimethoxytrityl group, p is 0 or 2, q is 8, L5 is a single bond, L6 is - (C = O) -NH-, and L5 is attached to the benzene ring in the para position, preferably where Tr is a 4-methoxytrityl group;

(30) A process for producing a compound represented by the formula (XI), the compound being a polynucleotide comprising a polynucleotide with a direct sense strand corresponding to a target gene, and a polynucleotide with an opposite direction strand having a polynucleotide sequence complementary to that of the direct sense strand polynucleotide, in which the 5 'end of the opposite strand polynucleotide and the 3' end of the direct strand strand polynucleotide are linked by X by phosphodiester bonds:

[Formula 4]

OO

, 11 II 1

HO-W2'-0-P — O — X — PO — W ^ -V-OH (XI)

OH OH

wherein W2 'represents a straight-stranded polynucleotide without hydroxy groups at the 5' end and the 3 'end; W1'-Y 'represents a counter-stranded polynucleotide without hydroxy groups at the 5' end and the 3 'end; and X represents the formula (XII):

[Formula 5]

image9

in which p represents an integer from 0 to 4; x represents an integer from 4 to 10; L5 represents a simple bond or -O-; L6 represents - (C = O) -NH- or -NH- (C = O) - starting from the link with (CH2) p; L5 is attached to the benzene ring in the meta or para position; provided that L5 is -O-, then, p represents an integer from 1 to 4; and the methylene group at the end is attached to the 3 'end of the straight-stranded polynucleotide to form a phosphodiester bond; and the oxygen atom attached to the phenyl group is attached to the 5 'end of the opposite strand polynucleotide to form a phosphodiester bond, the process comprising the steps of:

(i) reacting the hydroxy group of a compound represented by the formula Tr-OXH [in which Tr represents a protecting group of the hydroxy group, - (CH2) q- in X is attached to Tr-O- and the atom of Oxygen attached to the phenyl group is linked to hydrogen] with a compound represented by the formula (XIII):

[Formula 6]

or the formula (XIV):

image10

[Formula 7]

image11

[wherein R4 represents a 2-cyanoethyl group, a methyl group, a methanesulfonylethyl group, a 2,2,2-trichlorethyl group, or a 4-chlorophenylmethyl group, and R5 represents a morpholino group, a diisopropylamino group, a diethylamino group, or a dimethylamino group] to produce a compound represented by the formula (XV):

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[Formula 8]

R5

/

Tr-O-X-P.

O — R4

(XV)

(ii) reacting the compound obtained in step (i) with a compound represented by the formula HO-W1-Y-CPG [wherein W1-Y represents a counter-stranded polynucleotide protected without hydroxy groups at the end 5 'and the 3' end, and CPG represents a polymeric support having a linker capable of binding to the polynucleotide] according to a phosphoramidite process and subsequently producing a moiety represented by the formula Tr1-O-W2-OP (= O) ( OR4) -O- [in which Tr1 represents a protective group of the hydroxy group, and W2 represents a direct-strand polynucleotide protected without hydroxy groups at the 5 'end and the 3' end] according to a phosphoramidite process to produce a compound represented by the formula (XVI):

[Formula 9]

image12

; Y

(iii) split the compound obtained in step (ii) of the CPG and remove the protecting group;

(31) The process according to (30), in which Tr and Tr1 are, identically or differently, a 4- methoxytrityl group, a 4,4'-dimethoxytrityl group, a pixyl group, a trityl group, a levulinyl group, or a bis (trimethylsilyloxy) (cyclohexyloxy) silyl group;

(32) The process according to (30), in which Tr and Tr1 are, identically or differently, a 4- methoxytrityl group or a 4,4'-dimethoxytrityl group, the sum of pyq is an integer number of 4 or greater, L5 is a single bond, L6 is - (C = O) -NH-, and L5 is attached to the benzene ring in the para position, preferably where the sum of pyq is an integer of 8 or higher;

(33) The process according to (30), in which Tr and Tr1 are, identically or differently, a 4- methoxytrityl group or a 4,4'-dimethoxytrityl group, p is 0 or 2, which is a integer of 6 or greater, L5 is a single bond, L6 is - (C = O) -NH-, and L5 is attached to the benzene ring in the position for preferably where q is 6 or 8, more preferably in which is 8;

(34) The process according to (30), wherein Tr and Tr1 are, identically or differently, a 4- methoxytrityl group or a 4,4'-dimethoxytrityl group, p is 2, q is 8, L5 it is a simple bond, L6 is - (C = O) -NH-, and L5 is attached to the benzene ring in the para position, preferably where each Tr and Tr1 is a 4,4'-dimethoxytrityl group;

(35) The process according to any one of (30) to (34), wherein R4 is a 2-cyanoethyl group, a methyl group, a methanesulfonylethyl group, a 2,2,2-trichlorethyl group, or a 4-chlorophenylmethyl group, and R5 is a morpholino group, a diisopropylamino group, a diethylamino group, or a dimethylamino group, preferably wherein R4 is a 2-cyanoethyl group or a methyl group, and R5 is a morpholino group or a group diisopropylamino;

(36) The process according to any one of (30) to (34), in which the compound represented by the

formula (XIII) is chloro (morpholino) methoxyphosdine, chloro (morpholino) cyanoethoxyphosdine,

chloro (diisopropylamino) methoxyphosdine, or chloro (diisopropylamino) cyanoethoxyphosdine;

(37) The process according to any one of (30) to (34), wherein the compound represented by the formula (XIV) is bis (diisopropylamino) cyanoethoxyphosdine;

(38) A polynucleotide selected from the following, or a salt thereof: HO-Cp-Gm1p-Ap-Gm1p-Ap-Cm1p-Ap-

Um1p-Ap-Um1p-Gp-Gm1p-Gp-Um1p-Gp-Cm1p-Tp-Am1p-XP (= O) (OH) -O-Um1p-Tp-Am1p-Gp-Cm1p-Ap-Cm1p-Cp-Cm1p -Ap-Um1p- Gp-Um1p-Gp-Um1p-Cp-Um1p-Cp-Gm1p-Tp-Um1t-H (HS-005), HO-Cp-Am1p-Gp-Am1p-Cp-Am1p-Cp-Am1p- Tp-Gm1p-Gp-Gm1p-Tp- Gm1p-Cp-UrT | 1p-Ap-Um1p-XP (= O) (OH) -O-Um1p-Ap-Um1p-Ap-Gm1p-Cp-Am1p-Cp-Cm1p -Cp-Am1p-Tp-Gm1p-Tp-gm1p-Tp-Cm1p-

Tp-Gm1p-Tp-Um1t-H (HS-006), HO-Cp-Gm1p-Ap-Gm1p-Ap-Cm1p-Ap-Cm1p-Ap-Um1p-Gp-Gm1p-Gp-Um1p-Gp-Cm1p-Tp -Am1p-X- P (= O) (OH) -O-Um1p-Tp-Am1p-Gp-Cm1p-Ap-Cm1p-Cp-Cm1p-Ap-Um1p-Gp-Um1p-Gp-Um1p-Cp-Um1p- Cp-Gm1p-Ts-Um1t-H (HS- 005s), or, HO-Cp-Am1p-Gp-Am1p-Cp-Am1p-Cp-Am1p-Tp-Gm1p-Gp-Gm1p-Tp-Gm1p-Cp-Um1p -Ap-Um1p-XP (= O) (OH) -O-Um1p- Ap-Um1p-Ap-Gm1p-Cp-Am1p-Cp-Cm1p-Cp-Am1p-Tp-Gm1p-Tp-Gm1p-Tp-Cm1p- Tp-Gm1p-Ts-Um1 (-H (HS-006s)

in which each of Ap, Gp, Cp, Tp, Ts, Am1p, Gm1p, Cm1p, Um1p, and Um1t represents a nucleoside or a nucleotide having a structure represented by the following formula:

[Formula 10]

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image13

the sequence in the upstream direction of X represents a direct sense strand polynucleotide corresponding to a target gene; the sequence in the downstream direction of X represents a polynucleotide that has a counter-stranded polynucleotide that has a nucleotide sequence complementary to that of the stranded polynucleotide in the direct sense; X represents a linker that has a structure represented by the formula (XVII):

[Formula 11]

image14

the methylene group at the end is attached to the 3 'end of the straight-stranded polynucleotide to form a phosphodiester bond; and the oxygen atom attached to the phenyl group is attached to the 5 'end of the opposite strand polynucleotide to form a phosphodiester bond;

(39) A pharmaceutical composition comprising a polynucleotide or a salt thereof according to (38) as active ingredient;

(40) The pharmaceutical composition according to (39), in which the pharmaceutical composition is directed to the treatment of a disease derived from the expression of the Hsp47 gene, preferably fibrosis;

(41) A polynucleotide or salt thereof according to (38) for use in inhibiting the expression of the Hsp47 gene, in a mammal;

(42) A reagent comprising a polynucleotide or a salt thereof according to any one of (1) to (21) or (38).

Advantages of the invention

The present invention has provided a polynucleotide that has an effect of RNA interference and / or an inhibitory effect of gene expression. The present invention has also provided a polynucleotide that is resistant to at least one enzyme selected from RNAse, phosphatase, and exonuclease, and has an RNA interference effect and / or an gene expression inhibitory effect. The present invention has also provided a polynucleotide that is resistant to RNase, phosphatase, and exonuclease, and has an RNA interference effect and / or an inhibitory effect on gene expression. The present invention has also provided a polynucleotide that has an effect of RNA interference and / or an inhibitory effect of gene expression without the need for the step of separately producing a straight-stranded polynucleotide and a strand-shaped polynucleotide On the contrary and without the need of the complicated procedure to mix these strands with precision in the same quantities to form a double. The present invention allows the functional analysis of several genes using the

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polynucleotide, and provides a pharmaceutical composition comprising the polynucleotide.

The present invention has also provided a synthetic intermediate compound useful for obtaining the polynucleotide. The present invention has also provided a process for producing the polynucleotide.

Brief description of the drawings

[Figure 1] Figure 1 is a diagram showing the detail of step A-1.

[Figure 2] Figure 2 is a diagram showing the details of Procedures C and D.

[Figure 3] Figure 3 is a diagram showing the details of Procedures E and F.

[Figure 4] Figure 4 is a diagram showing the detail of Procedure G.

[Figure 5] Figure 5 is a diagram showing the structures of the compounds described in Reference Examples 3 to 14, 17, and 21 to 23.

[Figure 6] Figure 6 is a diagram showing polynucleotides corresponding to the human p-catenin gene (from now on here, examples of combinations of polynucleotides as strands of direct and opposite directions will be shown in each diagram, for the symbols, the filled circle (•) represents an aDn, and the unfilled circle (O) represents a 2'-O-methyl RNA.The line between the filled circle and the unfilled circle represents a phosphodiester bond between the nucleosides In the diagram, p represents -P (= O) (OH) - When p is attached, a hydrogen atom from the final hydroxy group of the polynucleotide is removed.When the end of the polynucleotide is not attached, the end 3 'or 5' of the DNA or 2'-O-methyl RNA is an OH group.n represents the number of carbon atoms.The same applies to Figures 7 and 11. The nucleotide sequence of each polynucleotide is also shown in the diagram.

[Figure 7] Figure 7 is a diagram showing polynucleotides corresponding to the human p-catenin gene.

[Figure 8] Figure 8 is a diagram showing the gene inhibition activities of polynucleotides analyzed by real-time PCR.

[Figure 9] Figure 9 is a diagram showing the gene inhibition activities of polynucleotides analyzed by real-time PCR.

[Figure 10] Figure 10 is a diagram showing the structures of the compounds described in Reference Examples 24 to 31.

[Figure 11] Figure 11 is a diagram showing polynucleotides corresponding to the human p-catenin gene.

[Figure 12] Figure 12 is a diagram showing the gene inhibition activities of polynucleotides analyzed by real-time PCR.

[Figure 13] Figure 13 is a diagram showing polynucleotides corresponding to the human p-catenin gene. For the symbols, the unfilled square (□) represents an RNA, the filled circle (•) represents a DNA, and the unfilled circle (o) represents a 2'-O-methyl RNA. The line between the nucleosides represents a phosphodiester bond. In the diagram, p represents -P (= O) (OH) -. When p is attached, a hydrogen atom from the final hydroxy group of the polynucleotide is removed. When the end of the polynucleotide is not attached, the 3 'or 5' end of the RNA, the DNA, or the 2'-O-methyl RNA is an OH group. n represents the number of carbon atoms. The same applies to Figures 15, 16, and 19. The nucleotide sequence of each polynucleotide is also shown in the diagram.

[Figure 14] Figure 14 is a diagram showing the gene inhibition activities of polynucleotides analyzed by real-time PCR.

[Figure 15] Figure 15 is a diagram showing polynucleotides corresponding to the mouse PKR gene. [Figure 16] Figure 16 is a diagram showing polynucleotides corresponding to the human Hsp47 gene. In the diagram, s represents a phosphorothioate bond.

[Figure 17] Figure 17 is a diagram showing the gene inhibition activities of polynucleotides analyzed by real-time PCR. The unfilled bar represents a polynucleotide concentration of 0.1 nM. The filled bar represents a polynucleotide concentration of 1 nM. The same applies to Figures 18 and 20.

[Figure 18] Figure 18 is a diagram showing the gene inhibition activities of polynucleotides analyzed by real-time PCR.

[Figure 19] Figure 19 is a diagram showing polynucleotides corresponding to the human Hsp47 gene. [Figure 20] Figure 20 is a diagram showing the gene inhibition activities of polynucleotides analyzed by real-time PCR.

Description of the realizations

1. Description of terms

In the present specification, the "target gene" is not especially limited on condition that it is RNA from cells, tissues, or individuals, in which or in which this gene is introduced (hereinafter, can be referred to as "receivers"). The target gene can be mRNA that translates to a protein or it can be non-coding aRn that does not translate to a protein. Examples of non-coding RNA include functional RNA, for example, an untranslated region of mRNA, tRNA, rRNA, non-coding RNA-like RNA (mRNA analogue mRNA), long non-coding RNA (long RNA), small nuclear RNA ( RRNA), nucleolar RNA

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small (RNA), and microRNA (miRNA). Specifically, the target gene can be endogenous to the receptors of the introduction, or it can be exogenous and introduced into it by an approach such as gene transfer. It can also be a gene present on a chromosome or an extrachromosomal gene. Examples of the exogenous gene include, but are not limited to, those derived from viruses, bacteria, fungi, and protozoa, which can infect receptors. The gene's function may be known or unknown.

Examples of said target gene may include genes whose expression is specifically increased and / or that are specifically mutated in patients who have a particular disease. Examples of diseases may include a disease of the central nervous system (for example, Alzheimer's disease, dementia, and eating disorders), inflammatory disease (for example, allergy, rheumatism, osteoarthritis and lupus erythematosus), cardiovascular disease (for example , hypertension, cardiomegaly, angina pectoris, arteriosclerosis, and hypercholesterolemia), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, pancreas cancer, liver cancer, bladder cancer, cancer of breast, cervical cancer, colorectal cancer, colon cancer, and rectal cancer), respiratory disease (e.g., pneumonia, bronchitis, asthma, and chronic obstructive pulmonary disease), diabetes mellitus, diabetic retinopathy, diabetic nephropathy, anemia ( for example, anemia associated with chronic disease and iron-resistant iron deficiency anemia), related macular degeneration wave with age, immune disease (for example, Crohn's disease, atopic dermatitis, autoimmune disease, immunodeficiency, and leukemia), liver / gallbladder disease (for example, non-alcoholic steatohepatitis, liver cirrhosis, hepatitis, liver failure, cholestasis , and stones), gastrointestinal disease (for example, an ulcer, enteritis, and hypoabsorption), infection, adipositis, and fibrosis (pulmonary fibrosis, liver fibrosis, renal fibrosis, myelofibrosis, etc.). Examples of the genes causing these diseases may include, but are not limited to, spindle kinesin protein (KSP), vascular endothelial growth factor, (VEGF), transthyretin (TTR), proprotein subtilisin convertase / kexin type 9 (PCSK9 ), pole type kinase (PLK), ApoB-100, M2 subunit of ribonucleotide reductase (RRM2), clusterin, heat shock protein 27 (Hsp27), survivin, eukaryotic-4E initiation factor (eIF-4E), molecule intercellular adhesion 1 (ICAM-1), the alpha subunit of interleukin 4 receptor (IL-4R-alpha), Factor XI, Factor VII, N-ras, H-ras, K-ras, bcl-2, bcl-xL , Her-1, Her-2, Her-3, Her-4, MDR-1, human p-catenin gene, DDX3 sequence polypeptide 3 (DEAD (Asp-Glu-Ala-Asp), bound to X) , sequence 1 of the myeloid cell leukemia gene (MCL1), PKR (Eif2ak2), Hsp47 (Serpinhl), Hepcidin, active protein c (APC), signal transducer and transcription activator (STAT3).

In the present specification, the "natural nucleoside" refers to a 2'-deoxynucleoside such as 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, 2'-deoxy-5-methylcytidine, and thymidine or a ribonucleoside such as adenosine, guanosine, citidine, 5-methylcytidine, and uridine. In addition, the "oligonucleotide" refers to an oligonucleotide consisting of a compound in which the sugar moiety of the nucleoside forms an ester with phosphoric acid. In the present specification, the terms "oligonucleotide" and "polynucleotide" are used interchangeably.

In the present specification, 2'-deoxyadenosine can be referred to as A1; 2'-deoxyguanosine can be referred to as G ‘; 2'-deoxycytidine can be referred to as C ‘; 2'-deoxy-5-methylcytidine can be referred to as 5meC ‘; Thymidine can be referred to as T ‘; 2'-deoxyuridine can be referred to as U ‘; Adenosine can be referred to as Art; guanosine can be referred to as Grt; Citidine can be referred to as Crt; 5- methylcitidine can be referred to as 5meCrt; and uridine can be referred to as Urt In addition, herein, the 2'-deoxyadenosine nucleotide can be referred to as Ap; the 2'-deoxyguanosine nucleotide can be referred to as Gp; the 2'-deoxycytidine nucleotide can be referred to as Cp; the 2'-deoxy-5-methylcytidine nucleotide can be referred to as 5meCp; a thymidine nucleotide can be referred to as Tp; a 2'-deoxyuridine nucleotide can be referred to as Up; adenosine nucleotide can be referred to as Arp; guanosine nucleotide can be referred to as Grp; Citidine nucleotide can be referred to as Crp; 5-methylcytidine nucleotide can be referred to as 5meCrp; and a uracil nucleotide can be referred to as Urp.

In the present specification, where there is a phosphorothioate ester form instead of a nucleotide phosphoester form, a counterpart of Ap can be referred to as As; a counterpart of Gp can be referred to as Gs; a counterpart of Cp can be referred to as Cs; a 5meCp counterpart can be referred to as 5meCs; a counterpart of Tp can be referred to as Ts; a Up counterpart can be referred to as Us; an Arp counterpart can be referred to as Ars; a Grp counterpart can be referred to as Grs; a Crp counterpart can be referred to as Crs; a 5meCrp counterpart can be referred to as 5meCrs; and a counterpart of Urp can be referred to as Urs.

In the present specification, the term "sugar modified nucleoside" refers to a nucleoside whose sugar moiety has been modified.

In particular, examples of 2'-O-methyl modification include 2'-O-methylnucleoside and 2'-O-methylnucleotide; an Art counterpart can be referred to as Am1t; a counterpart of Grt can be referred to as Gm1t; a counterpart of Crt can be referred to as Cm1t; a 5meCrt counterpart can be referred to as 5meCm1t; a counterpart of Urt can be referred to as Um1t; an Arp counterpart can be referred to as Am1p; a Grp counterpart can be referred to as Gm1p; a Crp counterpart can be referred to as Cm1p; a

5meCrp counterpart can be referred to as 5meCm1p; a counterpart of Urp can be referred to as Um1p; A counterpart of Ars can be referred to as Am1s; a counterpart of Gre can be referred to as Gm1s; a counterpart of Crs can be referred to as Cm1s; a counterpart of 5meCs can be referred to as 5meCm1s; and a counterpart of Urs can be referred to as Um1s.

5 In the present specification, the 2'-O, 4'-C-ethylene nucleotide unit and the "ENA unit" refer to those nucleosides that have an ENA, and also refer to nucleosides and nucleotides that have a ENA unit: a counterpart of A1 can be referred to as A2t; a counterpart of Ap can be referred to as Ae2p; a counterpart of As can be referred to as Ae2s; a counterpart of Gt can be referred to as G2t; a Gp counterpart can be referred to as Ge2p; a counterpart of Gs can be referred to as Ge2s; a counterpart of 5meCt can be referred to as C2t; a 5meCp counterpart can be referred to as Ce2p; a

5meCs counterpart can be referred to as Ce2s; a counterpart of Tt can be referred to as T2t; a counterpart of Tp can be referred to as Te2p; and a counterpart of Ts can be referred to as Te2s.

In the present specification, the nucleotide unit of 2'-O, 4'-C-methylene and the "unit 2 ', 4'-BNA / LNA" refer to those nucleosides and nucleotides having a 2', 4 '-BNA / LNA and also refer to nucleosides and 15 nucleotides that have a 2', 4'-BNA / LNA unit: a counterpart of At can be referred to as A1t; a counterpart of Ap can be referred to as Ae1p; a counterpart of As can be referred to as Ae1s; a counterpart of Gt can be referred to as G1; a Gp counterpart can be referred to as Ge1p; a counterpart of Gs can be referred to as Ge1s; a 5meCt counterpart can be referred to as C1t; a 5meCp counterpart can be referred to as Ce1p; a counterpart of 5meCs can be referred to as Ce1s; 20 a counterpart of Tt can be referred to as T1t; a counterpart of Tp can be referred to as Te1p; and a counterpart of Ts can be referred to as Te1s.

From now on in this document, the structural formula of each nucleotide is shown.

[Formula 12]

Claims (41)

5 10 fifteen twenty 25 30 35 1. A polynucleotide or salt thereof, the polynucleotide comprising a polynucleotide with a direct sense strand corresponding to a target gene, and a polynucleotide with an opposite strand having a polynucleotide sequence complementary to that of the sense stranded polynucleotide Direct and having a structure represented by the following formula, in which the 5 'end of the strand polynucleotide in the opposite direction and the 3' end of the strand polynucleotide in the direct direction are linked by a linker through phosphodiester bonds: [Formula 2] image 1 Po inuc eotido with strand in direct sense (CH2) Sense strand polynucleotide -5 contrary in which p represents an integer from 0 to 4, q represents an integer from 4 to 10, L5 represents a simple link or -O-, L6 represents - (C = O) -NH- or -NH- (C = O) - starting from the link with (CH2) p, L5 is attached to the benzene ring in the meta or para position, and provided that if L5 is -O-, then, p represents an integer from 1 to 4. 2. The polynucleotide or salt thereof according to claim 1, wherein the sum of pyq is an integer equal to 4 or greater, L5 is a single bond, L6 is - (C = O) -NH- , and L5 is attached to the benzene ring in the para position, preferably in which the sum of pyq is an integer equal to 8 or greater. 3. The polynucleotide or salt thereof according to claim 1, wherein p is 0 or 2, q is an integer equal to 6 or greater, L5 is a single bond, L6 is - (C = O) -NH-, and L5 is attached to the benzene ring in the position for, preferably where q is 6 or 8, more preferably where q is 8. 4. The polynucleotide or salt thereof according to claim 1, wherein p is 2, q is 8, L5 is a single bond, L6 is - (C = O) -NH-, and L5 is attached to the Benzene ring in position for. 5. A polynucleotide or salt thereof, the polynucleotide comprising a polynucleotide with a direct sense strand corresponding to a target gene, and a polynucleotide with an opposite strand having a polynucleotide sequence complementary to that of the polynucleotide with strand direct sense, in which the 5 'end of the strand polynucleotide in the opposite direction and the 3' end of the strand polynucleotide in the direct direction are joined by a linker to form a phosphodiester structure at any of said ends, the linker having a structure represented by the following formula: [Formula 1] image2 in which the oxygen atom attached to the phenyl group is attached to the 5 ′ end of the opposite strand to form a phosphodiester structure; any one of R1, R2, and R3 represents a structure represented by the following formula: 5 10 fifteen twenty 25 30 35 40 Four. Five fifty L1- (CH2) m-L2-LMCH2CH2o) ni- (CH2) n2-0-> in which m represents an integer from 0 to 4, n1 represents an integer from 0 to 4, n2 represents 0 or an integer from 2 to 10, L1 represents a simple link or -O-, L2 represents a single bond or -CH (-NH-L4-R) -, L3 represents a simple link, - (C = O) -NH-, or -NH- (C = O) - starting from the link with L2, provided that if L3 is not a single link, then n2 represents an integer from 2 to 10, provided that if each of L1 and L2 is a single bond, m is 1, and each of n1 and n2 is 0, then L3-O ^ represents -CH (COOH) NH- ( amino acid residue) j-Ser, -CH (COOH) NH- (amino acid residue) j-Thr, -CH (NH2) CO- (amino acid residue) j-Ser, or -CH (NH2) CO- (amino acid residue) j-Thr, in which the remainder of the hydroxy group of this serine or threonine is linked to the phosphate group of the 3 'end of the straight-stranded polynucleotide, and the amino group of the serine or threonine can be further substituted by an acyl group, j represents an integer from 0 to 2, L4 represents a single bond, a methylene group, a polymethylene group having 2 to 4 carbon atoms, or the structure - (C = O) -CH2-CH2- (C = O) -O- in which a group carbonyl of the structure - (C = O) -CH2-CH2- (C = O) - O- is attached to the amino group on the left end of the structural formula to form the structure -NH- (C = O) -CH2- CH2- (C = O) -O-, and R represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, a saturated or unsaturated carbonyl hydrocarbon group having 2 to 30 carbon atoms, or a saturated or unsaturated hydrocarbonoxycarbonyl group having 2 at 30 carbon atoms; Y the remaining two of R1, R2, and R3 each independently represent a group selected from the group consisting of a hydrogen atom, an alkyl group having 1 to 8 carbon atoms that a substituent may have, an alkoxy group having 1 to 8 carbon atoms that a substituent may have, a halogen atom, an alkylcarbonylamino group having an alkyl group having 1 to 9 carbon atoms, and an alkylcarbonyl group containing an alkyl group having 1 to 8 carbon atoms that can have a substituent 6. The polynucleotide or salt thereof according to claim 5, wherein each R1 and R3 is a hydrogen atom. 7. The polynucleotide or salt thereof according to claim 6, wherein each L1 and L2 is a single bond, L3 is - (C = O) -NH-, and the sum of m and n2 is an integer equal to 3 or greater, preferably 8 or greater. 8. The polynucleotide or salt thereof according to claim 6, wherein each L1 and L2 is a single bond, L3 is - (C = O) -NH-, m is 0 or 2, and n2 is a integer equal to 6 or greater, preferably 6 or 8, more preferably 8. 9. The polynucleotide or salt thereof according to claim 5, wherein each R1 and R3 is a hydrogen atom, each L1 and L2 is a single bond, L3 is - (C = O) -NH-, m is 2, and n2 is 8. 10. The polynucleotide or salt thereof according to any one of claims 1 to 9, wherein the direct-strand polynucleotide consists of a polynucleotide represented by the following formula (II), the counter-stranded polynucleotide It consists of a polynucleotide represented by the following formula (III), and the polynucleotide also has the following characteristics of (a) to (d): 5 '- (y-P) 9-y-Xt-3 ’(ii) Y image3 (a) and represents an RNA, p represents a 2'-OMeRNA, and each A and u represents a DNA; (b) t and u represent, identically or differently, any integer from 0 to 5; (c) (Y-P) 9-Y in the polynucleotide represented by formula (II) has a nucleotide sequence identical to that of the target gene; Y (d) (Y-P) 9-Y in formula (II) and p- (Y-p) 9 in formula (III) have complementary nucleotide sequences between 5 10 fifteen twenty 25 30 35 40 Four. Five yes. 11. The polynucleotide or salt thereof according to any one of claims 1 to 9, wherein the straight-stranded polynucleotide consists of a polynucleotide represented by the following formula (IV), the counter-stranded polynucleotide It consists of a polynucleotide represented by the following formula (V), and the polynucleotide also has the following characteristics of (a) to (d): S'-ía-píg-Op - ^ - S '(IV) Y 5 '"5S ~ (a-P) 9-du-3' (V), (a) a and p represent, differently, a DNA or a 2'-OMeRNA, 8 and A represent, identically or differently, a DNA or a 2'-OMeRNA, and u represents, in an identical or different way, any nucleotide selected from a DNA, an RNA, and a 2'-oMeRNA; (b) p represents an integer of 0 or 1, t is 0 when p is 0 and represents any integer from 0 to 5 when p is 1, s represents any integer of 0 or 1, and u represents any integer of 0 to 5; (c) (a-p) g-ap in the polynucleotide represented by formula (IV) has a nucleotide sequence identical to that of the target gene; Y (d) (a-p) g in formula (IV) and (a-p) g in formula (V) have nucleotide sequence complementary to each other. 12. The polynucleotide or salt thereof according to any one of claims 1 to 9, wherein the straight-stranded polynucleotide consists of a polynucleotide represented by the following formula (VI), the counter-stranded polynucleotide It consists of a polynucleotide represented by the following formula (VII), and the polynucleotide also has the following characteristics of (a) to (d): image4 Y image5 (a) a and p represent, differently, a DNA or a 2'-OMeRNA, 8 and A represent, identically or differently, a DNA or a 2'-OMeRNA, and u represents, in an identical or different way, any nucleotide selected from a DNA, an RNA, and a 2'-OMeRNA; (b) p represents an integer of 0 or 1, t is 0 when p is 0 and represents any integer from 0 to 5 when p is 1, s represents any integer of 0 or 1, and u represents any integer of 0 to 5; (c) p- (a-p) 8-ap in the polynucleotide represented by formula (VI) has a nucleotide sequence identical to that of the target gene; Y (d) (a-p) 8 in formula (VI) and (a-p) 8 in formula (VII) have complementary nucleotide sequences to each other. 13. The polynucleotide or salt thereof according to claim 11 or 12, wherein a is a DNA, and p is a 2'-OMeRNA. 14. The polynucleotide or salt thereof according to any one of claims 10 to 13, wherein At and Uu are, identically or differently, any of: DNA having a thymine base, an adenine base, or a guanine base; or 2'-OMeRNA having a uracil base, an adenine base, or a guanine base. 15. The polynucleotide or salt thereof according to any one of claims 10 to 14, wherein t is 0, and u is 2. 16. The polynucleotide or salt thereof according to any one of claims 11 to 14, wherein p and t are 0, s is 1, and u is 2. 17. The polynucleotide or salt thereof according to any one of claims 11 to 14, wherein p and t are 0, s is 0 or 1, u is 2, and U2 is a DNA or a 2'-OMeRNA. 18. The polynucleotide or salt thereof according to any one of claims 1 to 9, wherein the straight-stranded polynucleotide consists of a polynucleotide represented by the following formula (VIII), the counter-stranded polynucleotide It consists of a polynucleotide represented by the following formula (IX), and the polynucleotide also has the following characteristics of (a) to (c): 5 ’- (a-p) 9-3 T (VIII) 5 10 fifteen twenty 25 Y 5'-p- (a-p) 9- (a-p) -3 ’(IX), (a) a is a DNA, and p is a 2'-OMeRNA; (b) p- (a-p) g in the polynucleotide represented by formula (IX) has a nucleotide sequence complementary to that of the target gene; Y (c) (a-p) g in formula (VIII) and (a-p) g in formula (IX) have nucleotide sequences complementary to each other. 19. The polynucleotide or salt thereof according to any one of claims 10 to 18, wherein any or all of the 1 to 4 2'-OMeRNA moieties are substituted by an ENA or a 2 ', 4'-BNA / LNA 20. The polynucleotide or salt thereof according to any one of claims 10 to 19, wherein any or all of 1 to 4 DNA residues are substituted by an RNA, an ENA or a 2 ', 4'- BNA / LNA. 21. The polynucleotide or salt thereof according to any one of claims 1 to 20, wherein the nucleotides are linked together by a phosphodiester or phosphorothioate bond. 22. A pharmaceutical composition comprising a polynucleotide or a salt thereof according to any one of claims 1 to 21 as active ingredient. 23. The pharmaceutical composition according to claim 22, wherein the pharmaceutical composition is directed to the treatment of a disease derived from gene expression. 24. A polynucleotide or salt thereof selected from claims 1 to 21 for use in inhibiting the expression of a target gene in a mammal. 25. A compound represented by the formula (X) or a salt thereof: [Formula 3] image6 wherein Tr represents a hydroxy group protecting group; p represents an integer from 0 to 4; q represents an integer from 4 to 10; L5 represents a simple bond or -O-; L6 represents - (C = O) -NH- or -NH- (C = O) - starting from the bond with (CH2V; and L5 is attached to the benzene ring in the meta or para position; and it is characterized in that the compound It is not: image7 in which n is 4, 6 or 8; 5 image8 in which n is 3, 5 or 7; image9 in which n is 3, 5 or 7; or image10 26. The compound or salt thereof according to claim 25, wherein Tr is a 4-methoxytrityl group, a 4,4'-dimethoxytrityl group, a pixyl group, a trityl group, a levulinyl group, or a group bis (trimethylsilyloxy) (cyclohexyloxy) silyl. The compound or salt thereof according to claim 25, wherein Tr is a 4-methoxytrityl group or a 4,4'-dimethoxytrityl group, the sum of pyq is an integer equal to 4 or greater, L5 is a single bond, L6 is - (C = O) -NH-, and L5 is attached to the benzene ring in the position for, preferably in which the sum of pyq is an integer equal to 8 or greater. 28. The compound or salt thereof according to claim 25, wherein Tr is a 4-methoxytrityl group or a 5 10 fifteen twenty 25 30 35 4,4'-dimethoxytrityl group, p is 0 or 2, q is an integer equal to 6 or greater, L5 is a single bond, L6 is - (C = O) - NH-, and L5 is attached to the ring of benzene in the position for, preferably where q is 6 or 8. 29. The compound or salt thereof according to claim 25, wherein Tr is a 4-methoxytrityl group or a 4,4'-dimethoxytrityl group, p is 0 or 2, q is 8, L5 is a single bond , L6 is - (C = O) -NH-, and L5 is attached to the benzene ring in the para position, preferably where Tr is a 4-methoxytrityl group. 30. A method of producing a compound represented by the formula (XI), the compound being a polynucleotide comprising a polynucleotide with a direct sense strand corresponding to a target gene, and a polynucleotide with an opposite direction strand having a polynucleotide sequence complementary to that of the direct sense strand polynucleotide, in which the 5 'end of the opposite strand polynucleotide and the 3' end of the direct strand strand polynucleotide are linked by X by phosphodiester bonds: [Formula 4]
OO
, 11 II „ ho-w2, -o-p-o-x — P-O — W-V-OH (XI)
OH OH wherein W2 'represents a straight-stranded polynucleotide without hydroxy groups at the 5' end and the 3 'end; W1'-Y 'represents a counter-stranded polynucleotide without hydroxy groups at the 5' end and the 3 'end; and X represents the formula (XII): [Formula 5] image11 in which p represents an integer from 0 to 4; q represents an integer from 4 to 10; L5 represents a simple bond or -O-; L6 represents - (C = O) -NH- or -NH- (C = O) - starting from the link with (CH2) p; L5 is attached to the benzene ring in the meta or para position; provided that if L5 is -O-, then, p represents an integer from 1 to 4; and the methylene group at the end is attached to the 3 'end of the straight-stranded polynucleotide to form a phosphodiester bond; and the oxygen atom attached to the phenyl group is attached to the 5 'end of the opposite strand polynucleotide to form a phosphodiester bond, the process comprising the steps of: (i) reacting the hydroxy group of a compound represented by the formula Tr-OXH [in which Tr represents a protecting group of the hydroxy group, - (CH2) q- in X is attached to Tr-O- and the atom of Oxygen attached to the phenyl group is linked to hydrogen] with a compound represented by the formula (XIII): [Formula 6] image12 or the formula (XIV): [Formula 7] image13 [wherein R4 represents a 2-cyanoethyl group, a methyl group, a methanesulfonylethyl group, a 2,2,2-trichlorethyl group, or a 4-chlorophenylmethyl group, and R5 represents a morpholino group, a diisopropylamino group, a 5 10 fifteen twenty 25 30 35 40 Four. Five diethylamino group, or a dimethylamino group] to produce a compound represented by the formula (XV): [Formula 8] R5 i Tr-OX-P, O — R4 (XV) (ii) reacting the compound obtained in step (i) with a compound represented by the formula HO-W1-Y-CPG [wherein W1-Y represents a counter-stranded polynucleotide protected without hydroxy groups at the end 5 'and the 3' end, and CPG represents a polymeric support having a linker capable of binding to the polynucleotide] according to a phosphoramidite process and subsequently producing a moiety represented by the formula Tr1-O-W2-OP (= O) ( OR4) -O- [in which Tr1 represents a protective group of the hydroxy group, and W2 represents a direct-strand polynucleotide protected without hydroxy groups at the 5 'end and the 3' end] according to a phosphoramidite process to produce a compound represented by the formula (XVI): [Formula 9] image14 Y (iii) split the compound obtained in step (ii) of the CPG and remove the protective group. 31. The method according to claim 30, wherein Tr and Tr1 are, identically or differently, a 4- methoxytrityl group, a 4,4'-dimethoxytrityl group, a pixyl group, a trityl group, a levulinyl group, or a bis (trimethylsilyloxy) (cyclohexyloxy) silyl group. 32. The method according to claim 30, wherein Tr and Tr1 are, identically or differently, a 4- methoxytrityl group or a 4,4-dimethoxytrityl group, the sum of pyq is an integer equal to 4 or greater , L5 is a simple bond, L6 is - (C = O) -NH-, and L5 is attached to the benzene ring in the para position, preferably where the sum of pyq is an integer equal to 8 or greater. 33. The method according to claim 30, wherein Tr and Tr1 are, identically or differently, a 4- methoxytrityl group or a 4,4'-dimethoxytrityl group, p is 0 or 2, q is an equal integer at 6 or greater, L5 is a single bond, L6 is - (C = O) -NH-, and L5 is attached to the benzene ring in the position for, preferably where q is 6 or 8, more preferably in the What is 8. 34. The method according to claim 30, wherein Tr and Tr1 are, identically or differently, a 4- methoxytrityl group or a 4,4'-dimethoxytrityl group, p is 2, q is 8, L5 is a bond simple, L6 is - (C = O) -NH-, and L5 is attached to the benzene ring in the para position, preferably where each Tr and Tr1 is a 4,4'-dimethoxytrityl group. 35. The process according to any one of claims 30 to 34, wherein R4 is a 2- cyanoethyl group, a methyl group, a methanesulfonylethyl group, a 2,2,2-trichlorethyl group, or a group 4- chlorophenylmethyl, and R5 is a morpholino group, a diisopropylamino group, a diethylamino group, or a dimethylamino group, preferably wherein R4 is a 2-cyanoethyl group or a methyl group, and R5 is a morpholino group or a diisopropylamino group. 36. The method according to any one of claims 30 to 34, wherein the compound represented by the formula (XIII) is chloro (morpholino) methoxyphosdine, chloro (morpholino) cyanoethoxyphosdine, chloro (diisopropylamino) methoxyphosdine, or chlorine ( diisopropylamino) cyanoethoxyphosdine. 37. The process according to any one of claims 30 to 34, wherein the compound represented by the formula (XIV) is bis (diisopropylamino) cyanoethoxyphosdine. 38. A polynucleotide selected from the following, or a salt thereof: HO-Cp-Gm1p-Ap-Gm1p-Ap-Cm1p-Ap-Cm1p- Ap-Um1p-Gp-Gm1p-Gp-Um1p-Gp-Cm1p-Tp-Am1p-XP (= O) (OH) -O-Um1p-Tp-Am1p-Gp-Cm1p-Ap-Cm1p-Cp-Cm1p-Ap -Um1p-Gp-Um1p- Gp-Um1p-Cp-Um1p-Cp-Gm1p-Tp-Um1t-H (HS-005), HO-Cp-Am1p-Gp-Am1p-Cp-Am1p-Cp-Am1p-Tp- Gm1p-Gp-Gm1p-Tp-Gm1p-Cp- Um1p-Ap-Um1p-XP (= O) (OH) -O-Um1p-Ap-Um1p-Ap-Gm1p-Cp-Am1p-Cp-Cm1p-Cp-Am1p -Tp-Gm1p-Tp-Gm1p-Tp-Cm1p-Tp-Gm1p-Tp- Um1t-H (HS-006), HO-Cp-Gm1p-Ap-Gm1p-Ap-Cm1p-Ap-Cm1p-Ap-Cm1p-Ap-Um1p-Gp-Um1p-Gp-Cm1p-Tp-Am1p-XP (= O) (OH) -O- Um1p-Tp-Am1p-Gp-Cm1p-Ap-Cm1p-Cp-Cm1p-Ap-Um1p-Gp-Um1p-Gp-Um1p-Cp-Um1p-Cp-Gm1p-Ts-Um1t- H (HS-005s), or, HO-Cp- Am1p-Gp-Am1p-Cp-Am1p-Cp-Am1p-Tp-Gm1p-Gp-Gm1p-Tp-Gm1p-Cp-Um1p-Ap-Um1p-XP (= O) (OH) -O-Um1p-Ap-Um1p-Ap-GtT | 1p-Cp- 5 10 fifteen Am1P-Cp-Cm1p-Cp-Am1p-Tp-Gm1p-Tp-Gm1p-Tp-Cm1p-Tp-Gm1p-Tp-Gm1t-Ts-Um1t-H (HS-006s), in which each Ap, Gp, Cp , Tp, Ts, Am1p, Gm1p, Cm1p, Um1p, and Um1t represents a nucleoside or nucleotide having a structure represented by the following formula: [Formula 10] image15 the sequence in the upstream direction of X represents a direct sense strand polynucleotide corresponding to a target gene; the sequence in the downstream direction of X represents a polynucleotide that has a counter-stranded polynucleotide that has a nucleotide sequence complementary to that of the stranded polynucleotide in the direct sense; X represents a linker that has a structure represented by the formula (XVII): [Formula 11] image16 the methylene group at the end is attached to the 3 'end of the straight-stranded polynucleotide to form a phosphodiester bond; and the oxygen atom attached to the phenyl group is attached to the 5 'end of the opposite strand polynucleotide to form a phosphodiester bond. 39. A pharmaceutical composition comprising a polynucleotide or salt thereof according to claim 38 as active ingredient. 40. The pharmaceutical composition according to claim 39, wherein the pharmaceutical composition is for the treatment of a disease derived from the expression of the Hsp47 gene, preferably fibrosis. 41. A polynucleotide or salt thereof according to claim 38 for use in inhibiting the expression of the Hsp47 gene, in a mammal. 42. A reagent comprising a polynucleotide or a salt thereof according to any one of claims 1 to 21 or 38. image17 image18 00 Cl R5 V ' Tr-O-X-H image19 R5 R5 one You-O-X-P ^ OR (2. 3) HO-W ^ -O-P — O-X— P-O — W1 -r-OH I I OH OH G-3 stage OR OR G-2 stage Tr’-O-V ^ -O -P — O -X — P-O — W'-Y-CPG image20 (24) £ U image21 Reference example 3 n = 4 Reference example 4 n = 6 Reference example 5 n = 8; oo oo image22 Reference example n = 3 Reference example 7 n = 5 Reference example n = 7 image23 Reference example 9 n = 3 Reference example 10 n = 5 Reference example 11 n = 7 image24 reference 12 n = 3 reference 13 n = 5 reference 14 n = 7 image25 í K flj Reference Example 17 ■ o / 'sV' , *> • a Reference example 23 What k AoSer <MMTr) -Gly.Tyr * 0 £ í image26 image27 AMMMMm & MMbP '° ^ AT ° *one I '• to H ITS CT-169 5 ’GCA CAA GAA UGG AUC ACA CT-157 UT CGU GUT CTU ACC UAG TGT U5 ’ CT-437: n = 4 CT-ÍS5- n = 6 s’GCA CAA GAA UGG AUC ACA CT-456: n = 8 3’UT CGU GUT CTU ACC UAG TGT U CT-446: n = 3 CT-447: n = 5 CT-448: n = 7 5’GCA CAA GAA UGG AUC ACA 3 'UT CGU GUT CTU ACC UAG TGT U CT-449: n = 3 CT-450: n = S CT-451: n = 7 5’GCA CAA GAA UGG AUC ACA 3’UT CGU GUT CTU ACC UAG TGT U CT-452: n = 3 CT-453: n = 5 CT-454: n = 7 and ~ > ^ M * mP - <> 3 - * - v wwwwwwwwkw * ° r CT-169 5 ’GCA CAA GAA UGG AUC ACA CT-157 UT CGU GUT CTU ACC UAG TGT U5 ’ CT-460 5'GCA CAA GAA UGG AUC ACA 3 ’UT CGU GUT CTU ACC UAG TGT U CT-461 5 ’GCA CAA GAA UGG AUC ACA 3 ’UT CGU GUT CTU ACC UAG TGT U CT-462: R3 = Me CT-463: R * = H Relative amount relative to KC image28 [g einÉ-j] image29 co co image30 Reference Example 24 image31 Reference Example 25 image32 Reference example 26 Reference example 27 image33 Reference Example 28 Reference Example 29 Reference Example 30 Reference Example 31 [Figure 10] CT-169 CT-157 5‘GCA CAA GAA UGG AUC ACA UT CGU GUT CTU ACC UAG TGT U5 ’ 4 i '■ M you CT-448: n = 7 CT-465 n = 9 5’GCA CAA GAA UGG AUC ACA 3 'UT CGU GUT CTU ACC UAG TGT U i — 1 i — 1 co c ™ 6n_9 5’GCA CAA GAA UGG AUC ACA 3’UT CGU GUT CTU ACC UAG TGT U 6 £££ 6 £ 6it £ i £ <í £ i £ ov ... =! J " cwwwrm7mw? m '‘- Xr ° 5’GCA CAA GAA UGG AUC ACA 3’UT CGU GUT CTU ACC UAG TGT U CT-468; X = Single bond CT-489: X = -CHjCHr 5’GCA CAA GAA UGG AUC ACA CT-470: X = -CHjO- 3’UT CGU GUT CTU ACC UAG TGT U nh, CT-471 image34 p "° ^ 7 ™ CT-10G 5’GCA CMGAAUGG AUCACAAUU CT-041 uu CGU GUU CUU ACC UAG UGü U5 ’ CT-472 5’GCA CAA GAA UGG AUC ACA AUU 3 ’UU CGU GUU CUU ACC UAG UGU U CT-001 5'GCA CAA GAA UGG AUC ACA ATT CT-005 rr CGU GUU CUU ACC UAG UGU U5 ’ CT-473 5’GCA CAA GAA UGG AUC ACAA 3 ’UU CGU GUU CUU ACC UAG UGU U co -saw % L AC % L + e Relative amount with respect to NC You H- % V c> í % or ** to or V or or ’Bo E3 □ □ a o o o o í3 b or Ij O CO ¡3 w 3 ~ = c PK-001 PK-002 5 'GCUC GÜCUAÜGACAAGUAAUU GUCGAGCAGAUACUGUUCAUU 5 ' 5 'GCTCGUCUAUGACAAGTA PK-O09 3, utcgagcagaüactgtucatu co CD WWWWWWWWím? I »> ^ s HS-002 HS-005 HS-OOS HS-005S HS-OOSs UGAGACACAUGGGUGCUAUTT TTACUCUGUGUACCCACGAUA 5 ' 4 '■ 5 'GAGACACAUGGGUGCUAUATT TTCUCUGUGUACCCACGAUAU 5' 5 'CGAGACACAUGGGUGCTA 3' UTGCUCUGUGÜACCCACGATU 5 'CAGACACATGGGT GCUAU 3 'UTGT CTGTGTACCCACGAÜAU 5 'CGAGACACAUGGGUGCTA 3' UTGCUCUGUGÜACCCACGATU 5 'CAGACACAT GGGTGCUAU 3 'UTGTCTGTGTACCCACGAUAU image35 SIHSP47C 5 'GGACAGGCCUCUACAACUATT TTCCUGUCCGGAGAUGUUGAU 5' HS-012 5 'C GACAGGCCUCUACAACU 3 'UTGCUGUCCGGAGAUGUTGAU image36